T cell-based cancer immunotherapy has typically relied on membrane-bound cytotoxicity enhancers such as chimeric antigen receptors expressed in autologous αβT cells. These approaches are limited by tonic signalling of synthetic constructs and costs associated with manufacture of bespoke patient products. γδT cells are an emerging alternative chassis for cellular therapy, possessing innate anti-tumour activity, potent antibody-dependent cytotoxicity (ADCC) and minimal alloreactivity. We present an immunotherapeutic platform technology built around the Vγ9Vδ2 γδT cell chassis, harnessing specific characteristics of this cell type and offering an allo-compatible means of delivering cellular therapy that recruits bystander immunity. We engineered γδT cells to secrete synthetic opsonins and stabilized IL15 (stIL15). Using GD2 as a model antigen we show how opsonin-secreting Vγ9Vδ2 (OPS-γδ) have enhanced cytotoxicity and also confer this benefit on lymphoid and myeloid bystander cells. Reflecting the secreted nature of the engineered efficacy modules, the entire product rather than just the gene-modified fraction exhibited enhanced activation and cytotoxic profiles, superior persistence and proliferative capacity even upon repeated tumour challenge. Secretion of stIL15 abrogated the need for exogenous cytokine supplementation during expansion and further mediated functional licensing of bystander NK cells. Compared to unmodified γδT cells, stIL15-OPS-γδ cells exhibited superior in-vivo control of subcutaneous tumour and persistence in the blood. stIL15-OPS-γδ cells were further efficacious in 3D patient-derived osteosarcoma models, where efficacy could be boosted with the addition of immunomodulatory aminobisphosphonate drug, zoledronic acid. Together the data identify stIL15-OPS-γδ cells as a novel allogeneic platform combining direct cytolysis with bystander activation to effect solid tumour control. One Sentence Summary Armoured, opsonin-secreting OPS-γδ cell immunotherapy is built on the innate strengths of the Vγ9Vδ2 cell chassis for allogeneic solid tumour targeting.

Introduction The stress-inducible 72 kDa heat shock protein (mHsp70) is known to be expressed on the plasma membrane of tumour cells, but not on the corresponding normal cells. Targeting of the mHsp70 might provide a promising strategy for theranostics. Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as a promising contrast agents for the molecular magnetic resonance imagning (MRI). Coating of nanoparticles surface with therapeutic ligands could provide an anti-cancer activity. Material and methods Synthesised nanocomplexes were conjugated with anti-Hsp70 tools (including monoclonal cmHsp70.1 antibodies (cmHsp70.1-SPIONs), granzyme B (GrB-SPIONs), tumour penetrating peptide TPP (TPP-SPIONs)) and in vitro analysed for specific targeting of mHsp70 in cancer cells. Diagnostic potential of nanoparticles for MRI was assessed in the orthotopic models of U87 glioblastoma in NMRI nu/nu mice, C6 glioma in rats, orthotopic H1339 lung cancer in immunodeficient nu/nu mice, GL261 glioma in C57/Bl6 mice. To increase the tumour accumulation of particles additionally an external magnetic field was applied. Theranostic potential of GrB-SPIONs (that have a pro-apoptotic activity) was assessed as a monotherapy or in combination with radiotherapy. Results and discussions Superparamagentic conjugates had a high contrast enhancing properties (NMR relaxivity studies) and specifically targeted Hsp70-positive tumour cells (as shown by confocal and electron microscopies, flow cytometry) in a dose-dependent manner. High-resolution MRI (11 T) on T2-weighted images demonstrated the retention of conjugates in the tumour site. Biodistribution analysis employing highly sensitive non-linear magnetic response measurements (NLR- M 2 ) confirmed the retention of particles in the tumour (that was further confirmed by histological studies). Application of the external magnetic field further enhanced accumulation of the nanocarriers in the tumour. Due to the pro-apoptotic potency of GrB-SPIONs conjugates the latter, when being systemically administered in tumor-bearing animals, not only targeted the Hsp70-positive tumours but also induced the delay of cancer progression (MRI volumometry) and increased the survival. Combination of GrB-SPIONs with radiotherapy further enhanced the anti-cancer activity of the conjugates. Conclusion Overall, the data showed that Hsp70-targeted superparamagnetic nanoparticles have a potential to be used for the early detection and therapy of the tumours.

Retrospective studies on male breast cancer (MBC) have suff ered from small numbers of cases available from any one centre; thus a signifi cant problem in eff ectively studying this disease is accruing suffi ciently large numbers to allow comparative analysis of biomarkers associated with response.Using a coordinated multicentre approach, we present the fi rst large-scale study to address the relevance of the expression of hormone receptors in MBC and female breast cancer (FBC) using immunohistochemistry combined with a novel bioinformatics approach.Following ethical approval, 523 archival blocks (260 MBCs and 263 matched FBCs) were obtained retrospectively.Tissue microarrays were constructed and sections stained for ERα, ERβ1, ERβ2, ERβ5, total PR, PRA, PRB and AR and typed using CK5/6, CK14, CK18 and CK19 by immunohistochemistry. Following scoring, a range of ordination techniques were conducted on the datasets including hierarchical clustering and principal component analysis (PCA) to determine the diff erential nature of infl uences and interactions between MBC and FBC.Luminal A subgroup (ERα + and/or PR + , HER2 -) was the most common phenotype in both sexes.Luminal B (ERα + and/or PR + , HER2 + ) was not seen in males, while basal-like tumours (ERα -, PR -, HER2 -, CK5/6 + ) were infrequent in both.Hierarchical clustering revealed common clusters between MBC and FBC including total PR-PRA-PRB and ERβ1/2 clusters.ERα occurred on distinct clusters between males and females.AR, ERβ1, ERβ2 and ERβ5 all existed on the same cluster but with a diff erent substructure, particularly around the positioning of AR.ERα associated with this cluster in the male but not the female group.PCA confi rmed that in both groups strong infl uences came from PR-PRA-PRB.In MBC strong infl uences additionally came from AR and ERβ1, ERβ2 and ERβ5, whereas in FBC strong infl uences came from ERα alone.Our data support the hypothesis that breast cancer is biologically diff erent in male and females, which could have implications for therapy. O2

Research groups developing antibody therapies generate diverse data sets; the value of these sets would be compounded when shared or amalgamated. A complete amalgamation of diverse data sets requires data standards for information collection during experiments. We propose to define elements of the data standards in the form of common data elements (CDEs) in order to clarify each experiment's targets and data values. We have created a set of core information elements which we suggest should be collected from antibody therapy experiments. We propose these as a basis for community consultation with a view to defining a set of data standards which can be developed under the auspices of the Antibody Society.

Introduction: Magnetic fluid hyperthermia (MFH) is a novel technique whereby superparamagnetic iron oxide nanoparticles (SPIONs) are placed within and excited by an alternating magnetic field to generate heat.1 This approach for cancer therapy has previously been demonstrated in patients using direct intratumoural injection of SPIONs.2,3 We propose MFH can be delivered more effectively using antibody-targeted SPIONs. To test this hypothesis we have developed a recombinant single chain antibody fragment (scFv) to deliver SPIONs to the epithelial restricted avß6 integrin, an exciting new target in head and neck cancers.4

Immunoconjugates of antibodies with toxic agents are becoming a significant component of anticancer treatments. One ongoing challenge to the success of this approach is the exposure of healthy tissues to the cytotoxic agents employed. We propose that magnetic fluid hyperthermia, using pre-targeted antibody-iron oxide magnetic nanoparticles, can address this issue. In magnetic fluid hyperthermia an alternating magnetic field is applied to magnetic nanoparticles in target tissues causing localised heating and cell death. The externally employed magnetic field can be focused on the tumour deposit. The system has potential to provide potent and selective cancer-targeted hyperthermic therapy.

Background Bevacizumab is an angiogenesis inhibitor and a new therapy for the treatment of colorectal cancer. It is a humanized monoclonal antibody that targets vascular endothelial growth factor. Methods This review is based on a literature search of Medline, Pubmed, ISI web of knowledge and other published work for original articles, reviews and abstracts relevant to the surgical management of colorectal cancer with bevacizumab. Results and conclusion Combined with current chemotherapy regimens, bevacizumab offers a significant survival advantage, making it likely to see widespread use. Despite being generally well tolerated, serious toxicities, including wound complications and gastrointestinal perforation, have been reported that affect surgical management. Consideration should be given to the timing of surgical and adjuvant intervention when using this drug.

MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody–antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins.

The 'magic bullet' concept predicted over a century ago that antibodies would be used to target cancer therapy. Since then initial problems that were related to specificity, purity and immungenicity of antibody-based reagents have slowly been overcome due to developments in technology and increased knowledge. As a result, antibodies are in use for many clinical applications and now comprise the second largest category of medicines in clinical development after vaccines. For antibody-based cancer therapeutics the last 20 years have met with an explosion of knowledge about the biology of the disease and potential targets as well as new technology which allows cloning and manipulation of multifunctional antibody-based molecules. However, the focus still remains on developing therapeutics that will have potential for treating cancer in people and this is efficiently assessed in mechanistic clinical trials that feed back to the laboratory for further development. This review illustrates the mechanistic approach to making new molecules for antibody imaging and therapy of cancer. It is illustrated by examples of radioimmunotherapy and antibody-directed enzyme prodrug therapy developed by the authors.

An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.

AbstractOne of the major limitations of using intact immunoglobulins for targeting tumors is the poor penetration into tissues. Although small single chain Fv (scFv) antibody fragments have been used because of their improved kinetics, some exhibit high renal accumulation. In this study we show a significant reduction of renal accumulation of His‐tagged scFv's directly labelled with 99mTc‐tricarbonyl by use of succinic anhydride. Further decrease of renal uptake will make this approach hopefully suitable for therapeutic application, using 188/186Re(I)‐tricarbonyl.

Antibody‐directed enzyme prodrug therapy (ADEPT) targets an enzyme selectively to a tumor where it converts a relatively non‐toxic prodrug to a potent cytotoxic drug. Previous clinical work using antibody‐enzyme chemical conjugates has been limited by the moderate efficiency of tumor targeting of these molecules. To address this a recombinant fusion protein composed of MFE‐23, an anti‐carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody, fused to the amino‐terminus of the enzyme carboxypeptidase G2 (CPG2) has been constructed to achieve ADEPT in CEA‐producing tumors. MFE‐23::CPG2 fusion protein was overexpressed in Escherichia coli and purified using CEA affinity chromatography. Efficacy of MFE‐23::CPG2 delivery to tumors in vivo was assessed by measuring catalytic activity after intravenous injection of purified MFE‐23::CPG2 into nude mice bearing CEA‐positive LS174T human colon adenocarcinoma xenografts. Recombinant MFE‐23::CPG2 cleared rapidly from circulation and catalytic activity in extracted tissues showed tumor to plasma ratios of 1.5:1 (6 hr), 10:1 (24 hr), 19:1 (48 hr) and 12:1 (72 hr). 125I‐MFE‐23::CPG2 was retained in kidney, liver and spleen but MFE‐23::CPG2 catalytic activity was not, resulting in excellent tumor to normal tissue enzyme ratios 48 hr after injection. These were 371:1 (tumor to liver), 450:1 (tumor to lung), 562:1 (tumor to kidney), 1,477:1 (tumor to colon) and 1,618:1 (tumor to spleen). Favorable tumor : normal tissue ratios occurred at early time points when there was still 21% (24 hr) and 9.5% (48 hr) of the injected activity present per gram of tumor tissue. The high tumor concentrations and selective tumor retention of active enzyme delivered by MFE‐23::CPG2 establish that this recombinant fusion protein has potential to give improved clinical efficiency for ADEPT. Int. J. Cancer 85:571–577, 2000. © 2000 Wiley‐Liss, Inc.